Combination therapy for the treatment of cancer

ABSTRACT

The present disclosure relates to a pharmaceutical combination comprising (1) a first agent which is a CDK inhibitor or a pharmaceutically acceptable salt thereof and (2) a second agent which is an anti-hormonal agent or a pharmaceutically acceptable salt thereof. The present disclosure also relates to a pharmaceutical combination comprising (1) a first agent which is a CDK inhibitor or a pharmaceutically acceptable salt thereof, (2) a second agent which is an anti-hormonal agent or a pharmaceutically acceptable salt thereof, and (3) a third agent which is an agent that regulates the PI3K/Akt/mTOR pathway or a pharmaceutically acceptable salt thereof.

FIELD OF THE DISCLOSURE

The present disclosure relates to a pharmaceutical combinationcomprising a CDK inhibitor and an anti-hormonal agent for the treatmentof cancer; the uses of such combinations in the treatment of cancer; andto a method of treating warm-blooded animals including humans sufferingcancer comprising administering to said animal in need of such treatmentan effective dose of a CDK inhibitor and an anti-hormonal agent. Inaddition, the combination can optionally include an agent that regulatesthe PI3K/Akt/mTOR pathway.

BACKGROUND OF THE DISCLOSURE CDK Inhibitors

Tumor development is closely associated with genetic alteration andderegulation of CDKs and their regulators, suggesting that inhibitors ofCDKs may be useful anti-cancer therapeutics. Indeed, early resultssuggest that transformed and normal cells differ in their requirementfor, e.g., cyclin D/CDK4/6 and that it may be possible to develop novelantineoplastic agents devoid of the general host toxicity observed withconventional cytotoxic and cytostatic drugs.

The function of CDKs is to phosphorylate and thus activate or deactivatecertain proteins, including e.g. retinoblastoma proteins, lamins,histone H1, and components of the mitotic spindle. The catalytic stepmediated by CDKs involves a phospho-transfer reaction from ATP to themacromolecular enzyme substrate. Several groups of compounds (reviewedin e.g. Fischer, P. M. Curr. Opin. Drug Discovery Dev. 2001, 4, 623-634)have been found to possess anti-proliferative properties by virtue ofCDK-specific ATP antagonism.

At a molecular level mediation of CDK/cyclin complex activity requires aseries of stimulatory and inhibitory phosphorylation, ordephosphorylation, events. CDK phosphorylation is performed by a groupof CDK activating kinases (CAKs) and/or kinases such as wee1, Myt1 andMik1. Dephosphorylation is performed by phosphatases such as cdc25(a &c), pp2a, or KAP.

CDK/cyclin complex activity may be further regulated by two families ofendogenous cellular proteinaceous inhibitors: the Kip/Cip family, or theINK family. The INK proteins specifically bind CDK4 and CDK6. p16ink4(also known as MTS1) is a potential tumour suppressor gene that ismutated, or deleted, in a large number of primary cancers. The Kip/Cipfamily contains proteins such as p21Cip1, Waf1, p27Kip1 and p57kip2,where p21 is induced by p53 and is able to inactivate theCDK2/cyclin(E/A) complex. Atypically low levels of p27 expression havebeen observed in breast, colon and prostate cancers. Conversely overexpression of cyclin E in solid tumours has been shown to correlate withpoor patient prognosis. Over expression of cyclin D1 has been associatedwith oesophageal, breast, squamous, and non-small cell lung carcinomas.

The pivotal roles of CDKs, and their associated proteins, inco-ordinating and driving the cell cycle in proliferating cells havebeen outlined above. Some of the biochemical pathways in which CDKs playa key role have also been described. The development of monotherapiesfor the treatment of proliferative disorders, such as cancers, usingtherapeutics targeted generically at CDKs, or at specific CDKs, istherefore potentially highly desirable. Thus, there is a continued needto find new therapeutic agents to treat human diseases.

Anti-Hormonal Agent

Anti-hormonal agent works in two ways: (1) by lowering the amount of thehormone in the body or (2) by blocking the action of hormone on cells.

Various types of anti-hormonal agents are known.

One type of anti-hormonal agents is known as aromatase inhibitors.Aromatase inhibitors work by inhibiting the action of the enzymearomatase, which converts androgens into estrogens by a process calledaromatization. As breast tissue is stimulated by estrogens, decreasingtheir production is a way of suppressing recurrence of the breast tumortissue. The main source of estrogen is the ovaries in premenopausalwomen, while in post-menopausal women most of the body's estrogen isproduced in peripheral tissues (outside the CNS), and also a few CNSsites in various regions within the brain. Estrogen is produced and actslocally in these tissues, but any circulating estrogen, which exertssystemic estrogenic effects in men and women, is the result of estrogenescaping local metabolism and spreading to the circulatory system. Thereare two types of aromatase inhibitors: (1) steroidal inhibitors, such asexemestane (Aromasin) which forms a permanent and deactivating bond withthe aromatase enzyme; and (2) non-steroidal inhibitors, such asanastrozole (Arimidex) or Letrozole (Femara) which inhibit the synthesisof estrogen via reversible competition for the aromatase enzyme.

Another type of anti-hormonal agent is estrogen receptor antagonist. Anexample of an estrogen receptor antagonist is fulvestrant (Faslodex).Estrogen receptors are found in and on breast cells. Estrogen binds toestrogen receptors, like a key fitting into a lock. This can activatethe receptor and cause hormone receptor-positive tumors to grow.Fulvestrant binds to and blocks estrogen receptors and reduces thenumber of estrogen receptors in breast cells.

Another type of anti-hormonal agent is selective estrogen receptormodulators (SERMs) are a class of compounds that act on the estrogenreceptor. A characteristic that distinguishes these substances from purereceptor agonists and antagonists is that their action is different invarious tissues, thereby granting the possibility to selectively inhibitor stimulate estrogen-like action in various tissues An example of aSERM is tamoxifen. Tamoxifen is an estrogen receptor agonist at bone anduterus, but an antagonist at breast.

Agent that Regulates the PI3K/Akt/mTOR Pathway

The PI3K/Akt/mTOR pathway is an important, tightly regulated survivalpathway for the normal cell. Phosphatidylinositol 3-kinases (PI3Ks) arewidely expressed lipid kinases that catalyze the transfer of phosphateto the D-3′ position of inositol lipids to producephosphoinositol-3-phosphate (PIP), phosphoinositol-3,4-diphosphate(PIP₂) and phosphoinositol-3,4,5-triphosphate (PIP₃). These products ofthe PI3K-catalyzed reactions act as second messengers and have centralroles in key cellular processes, including cell growth, differentiation,mobility, proliferation and survival.

Of the two Class 1 PI3Ks, Class 1A PI3Ks are heterodimers composed of acatalytic p110 subunit (α, β, δ isoforms) constitutively associated witha regulatory subunit that can be p85α, p55α, p50α, p85β or p55γ. TheClass 1B sub-class has one family member, a heterodimer composed of acatalytic p110γ subunit associated with one of two regulatory subunits,p101 or p84 (Fruman et al., Annu Rev. Biochem. 67:481 (1998); Suire etal., Curr. Biol. 15:566 (2005)).

In many cases, PIP2 and PIP3 recruit AKT to the plasma membrane where itacts as a nodal point for many intracellular signaling pathwaysimportant for growth and survival (Fantl et al., Cell 69:413-423(1992);Bader et al., Nature Rev. Cancer 5:921 (2005); Vivanco and Sawyer,Nature Rev. Cancer 2:489 (2002)). Aberrant regulation of PI3K, whichoften increases survival through AKT activation, is one of the mostprevalent events in human cancer and has been shown to occur at multiplelevels. The tumor suppressor gene PTEN, which dephosphorylatesphosphoinositides at the 3′ position of the inositol ring and in sodoing antagonizes PI3K activity, is functionally deleted in a variety oftumors. In other tumors, the genes for the p110a isoform, P/K3CA, andfor AKT are amplified and increased protein expression of their geneproducts has been demonstrated in several human cancers. Further,somatic missense mutations in P/K3CA that activate downstream signalingpathways have been described at significant frequencies in a widediversity of human cancers (Kang at el., Proc. Natl. Acad. Sci. USA102:802 (2005); Samuels et al., Science 304:554 (2004); Samuels et al.,Cancer Cell 7:561-573 (2005)). Thus, inhibitors of PI3K alpha are knownto be of particular value in the treatment of cancer and otherdisorders.

mTOR is a kinase protein predominantly found in the cytoplasm of thecell. It acts as a central regulator of many biological processesrelated to cell proliferation, angiogenesis, and cell metabolism. mTORexerts its effects primarily by turning on and off the cell'stranslational machinery, which includes the ribosomes, and isresponsible for protein synthesis. mTOR is a key intracellular point ofconvergence for a number of cellular signaling pathways. mTOR performsits regulatory function in response to activating or inhibitory signalstransmitted through these pathways, which are located upstream from mTORin the cell. These diverse signaling pathways are activated by a varietyof growth factors (including vascular endothelial growth factors(VEGFs), platelet-derived growth factor (PDGF), epidermal growth factor(EGF), insulin-like growth factor 1 (IGF-1)), hormones (estrogen,progesterone), and the presence or absence of nutrients (glucose, aminoacids) or oxygen. One or more of these signaling pathways may beabnormally activated in patients with many different types of cancer,resulting in deregulated cell proliferation, tumor angiogenesis, andabnormal cell metabolism.

In spite of numerous treatment options for cancer patients, thereremains a need for effective and safe therapeutic agents and a need fortheir preferential use in combination therapy.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to a pharmaceutical combinationcomprising (1) a first agent which is a CDK inhibitor or apharmaceutically acceptable salt thereof and (2) a second agent which isan anti-hormonal agent or a pharmaceutically acceptable salt thereof.

The present disclosure also relates to a pharmaceutical combinationcomprising (1) a first agent which is a CDK inhibitor or apharmaceutically acceptable salt thereof, (2) a second agent which is ananti-hormonal agent or a pharmaceutically acceptable salt thereof, and(3) a third agent which is an agent that regulates the PI3K/Akt/mTORpathway or a pharmaceutically acceptable salt thereof.

Such combination may be for simultaneous, separate or sequential use forthe treatment of a cancer.

In one embodiment, the CDK inhibitor is CDK4/6 inhibitor.

The CDK4/6 inhibitor can be, for example,

Compound A1, described by Formula A1 below:

or,

Compound A2, described by Formula A2 below:

or,

palbociclib (hereinafter referred as Compound A3, also known asPD-0332991).

Compound A1 is also described by the chemical name7-Cyclopentyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid dimethylamide.

Compound A2 is also described by the chemical name7-cyclopentyl-N,N-dimethyl-2-(5-((1R,6S)-9-methyl-4-oxo-3,9-diazabicyclo[4.2.1]nonan-3-yl)pyridin-2-ylamino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide.

Compound A3 is also described by the chemical name6-Acetyl-8-cyclopentyl-5-methyl-2-{[5-(1-piperazinyl)-2-pyridinyl]amino}pyrido[2,3-d]pyrimidin-7(8H)-one.

In one embodiment, the anti-hormonal agent is an aromatase inhibitor.Such aromatase inhibitor can be either a non-steroidal aromataseinhibitor or a steroidal aromatase inhibitor.

Letrozole (hereinafter referred as Compound 1) is an example of anon-steroidal aromatase inhibitor.

Exemestane (hereinafter referred as Compound B2) is an example of asteroidal aromatase inhibitor.

In another embodiment, the anti-hormonal agent is an estrogen receptorantagonist.

Fulvestrant (hereinafter referred as Compound B3) is an example of anestrogen receptor antagonist.

In yet another embodiment, the anti-hormonal agent is a selectiveestrogen receptor modulator.

Tamoxifen (hereinafter referred as Compound B4) is an example of aselective estrogen receptor modulator.

In one embodiment, the agent that regulates the PI3K/Akt/mTOR pathway isa PI3K inhibitor.

The PI3K inhibitor can be, for example,

Compound C1, described by Formula C1 below:

or,

Compound C2, described by Formula C2 below:

Compound C1 is also described by the chemical name(S)-Pyrrolidine-1,2-dicarboxylic acid 2-amide1-({4-methyl-5-[2-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-pyridin-4-yl]-thiazol-2-yl}-amide).

Compound C2 is also described by the chemical name5-(2,6-di-4-morpholinyl-4-pyrimidinyl)-4-(trifluoromethyl)-2-pyrimidinamine.

In another embodiment, the agent that regulates the PI3K/Akt/mTORpathway is a mTOR inhibitor.

Everolimus (hereinafter referred as Compound C3) is an example of a mTORinhibitor.

The present disclosure further relates to the above pharmaceuticalcombination(s) for use in the treatment of a cancer.

The present disclosure further relates to a method for the treatment ofa cancer comprising administering the above pharmaceuticalcombination(s) in jointly therapeutically effective amount, to awarm-blooded animal, preferably a human, in need thereof.

In accordance with the present disclosure, the compounds in thepharmaceutical combination(s) may be administered either as a singlepharmaceutical composition, as separate compositions, or sequentially.

In a specific embodiment, the disclosure relates to a pharmaceuticalcombination comprising (1) a first agent which is Compound A1 describedby Formula A1 below or a pharmaceutically acceptable salt thereof:

-   -   and (2) a second agent which is letrozole.

In another specific embodiment, the disclosure relates to apharmaceutical combination comprising (1) a first agent which isCompound A1 described by Formula A1 below or a pharmaceuticallyacceptable salt thereof:

-   -   (2) a second agent which is letrozole, and    -   (3) a third agent which is Compound C1, described by Formula C1        below or a pharmaceutically acceptable salt thereof:

In another specific embodiment, the disclosure relates to apharmaceutical combination comprising (1) a first agent which isCompound A1 described by Formula A1 below or a pharmaceuticallyacceptable salt thereof:

-   -   (2) a second agent which is letrozole, and    -   (3) a third agent which is Compound C2, described by Formula C2        below or a pharmaceutically acceptable salt thereof:

In another specific embodiment, the disclosure relates to apharmaceutical combination comprising (1) a first agent which isCompound A1 described by Formula A1 below or a pharmaceuticallyacceptable salt thereof:

-   -   and (2) a second agent which is fulvestrant.

In another specific embodiment, the disclosure relates to apharmaceutical combination comprising (1) a first agent which isCompound A1 described by Formula A1 below or a pharmaceuticallyacceptable salt thereof:

-   -   (2) a second agent which is fulvestrant, and    -   (3) a third agent which is Compound C1, described by Formula C1        below or a pharmaceutically acceptable salt thereof:

In another specific embodiment, the disclosure relates to apharmaceutical combination comprising (1) a first agent which isCompound A1 described by Formula A1 below or a pharmaceuticallyacceptable salt thereof:

-   -   (2) a second agent which is fulvestrant, and    -   (3) a third agent which is Compound C2, described by Formula C2        below or a pharmaceutically acceptable salt thereof:

In another specific embodiment, the disclosure relates to apharmaceutical combination comprising (1) a first agent which isCompound A1 described by Formula A1 below or a pharmaceuticallyacceptable salt thereof:

-   -   (2) a second agent which is everolimus, and    -   (3) a third agent which is exemestane.

In another specific embodiment, the disclosure relates to a method oftreating HR+, HER2− breast cancer comprising administering to a subjecta pharmaceutical combination comprising (1) a first agent which isCompound A1 described by Formula A1 below or a pharmaceuticallyacceptable salt thereof:

-   -   and (2) a second agent which is letrozole.

In another specific embodiment, the disclosure relates to a method oftreating ER+, HER2− advanced breast cancer comprising administering to asubject a pharmaceutical combination comprising (1) a first agent whichis Compound A1 described by Formula A1 below or a pharmaceuticallyacceptable salt thereof:

-   -   and (2) a second agent which is letrozole.

In another specific embodiment, the disclosure relates to a method oftreating ER+ advanced breast cancer comprising administering to asubject a pharmaceutical combination comprising (1) a first agent whichis Compound A1 described by Formula A1 below or a pharmaceuticallyacceptable salt thereof:

-   -   (2) a second agent which is letrozole, and    -   (3) a third agent which is Compound C1, described by Formula C1        below or a pharmaceutically acceptable salt thereof:

In another specific embodiment, the disclosure relates to a method oftreating ER+ advanced breast cancer comprising administering to asubject a pharmaceutical combination comprising (1) a first agent whichis Compound A1 described by Formula A1 below or a pharmaceuticallyacceptable salt thereof:

-   -   (2) a second agent which is letrozole, and    -   (3) a third agent which is Compound C2, described by Formula C2        below or a pharmaceutically acceptable salt thereof:

In another specific embodiment, the disclosure relates to a method oftreating postmenopausal woman with ER+, HER2− breast cancer comprisingadministering to a subject a pharmaceutical combination comprising (1) afirst agent which is Compound A1 described by Formula A1 below or apharmaceutically acceptable salt thereof:

-   -   and (2) a second agent which is fulvestrant.

In another specific embodiment, the disclosure relates to a method oftreating postmenopausal woman with ER+, HER2− breast cancer comprisingadministering to a subject a pharmaceutical combination comprising (1) afirst agent which is Compound A1 described by Formula A1 below or apharmaceutically acceptable salt thereof:

-   -   (2) a second agent which is fulvestrant, and    -   (3) a third agent which is Compound C1, described by Formula C1        below or a pharmaceutically acceptable salt thereof:

In another specific embodiment, the disclosure relates to a method oftreating postmenopausal woman with ER+, HER2− breast cancer comprisingadministering to a subject a pharmaceutical combination comprising (1) afirst agent which is Compound A1 described by Formula A1 below or apharmaceutically acceptable salt thereof:

-   -   (2) a second agent which is fulvestrant, and    -   (3) a third agent which is Compound C2, described by Formula C2        below or a pharmaceutically acceptable salt thereof:

In another specific embodiment, the disclosure relates to a method oftreating ER+ breast cancer comprising administering to a subject apharmaceutical combination comprising (1) a first agent which isCompound A1 described by Formula A1 below or a pharmaceuticallyacceptable salt thereof:

-   -   (2) a second agent which is everolimus, and    -   (3) a third agent which is exemestane.

The present disclosure further relates to a kit comprising thepharmaceutical combination.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 shows an extended dose matrix and isobologram demonstrating theeffects of combining Compound A1 and Compound 1 doses on proliferationof MCF7/ARO human breast carcinoma cells with Δ4A.

FIG. 2 shows an extended dose matrix and isobologram demonstrating theeffects of combining Compound A1 and Compound B2 doses on proliferationof MCF7/ARO human breast carcinoma cells with Δ4A.

FIG. 3 shows an extended dose matrix and isobologram demonstrating theeffects of combining Compound A1 and Compound B3 doses on proliferationof MCF7/ARO human breast carcinoma cells with Δ4A.

FIG. 4 shows an extended dose matrix demonstrating the effects ofcombining Compound A1 and Compound 1 with or without the presence ofCompound C1, Compound C2 or Compound C3 on proliferation of MCF7/AROhuman breast carcinoma cells with Δ4A.

FIG. 5 shows an extended dose matrix demonstrating the effects ofcombining Compound A1 and Compound B2 with or without the presence ofCompound C1, Compound C2 or Compound C3 on proliferation of MCF7/AROhuman breast carcinoma cells with Δ4A.

FIG. 6 shows an extended dose matrix demonstrating the effects ofcombining Compound A1 and Compound B3 with or without the presence ofCompound C1, Compound C2 or Compound C3 on proliferation of MCF7/AROhuman breast carcinoma cells with Δ4A.

FIG. 7 shows the MCF7/Aro Cell Growth for 6 Days w Δ4A with the CTGAssay.

FIG. 8 shows an extended dose matrix and isobologram demonstrating theeffects of combining Compound A3 and Compound 1 doses on proliferationof MCF7/ARO human breast carcinoma cells with Δ4A.

FIG. 9 shows an extended dose matrix and isobologram demonstrating theeffects of combining Compound A2 and Compound 1 doses on proliferationof MCF7/ARO human breast carcinoma cells with Δ4A.

FIG. 10 shows an extended dose matrix demonstrating the effects ofcombining Compound A3 and Compound 1 with or without the presence ofCompound C1 or Compound C3 on proliferation of MCF7/ARO human breastcarcinoma cells with Δ4A.

FIG. 11 shows an extended dose matrix demonstrating the effects ofcombining Compound A2 and Compound 1 with or without the presence ofCompound C1 or Compound C3 on proliferation of MCF7/ARO human breastcarcinoma cells with Δ4A.

FIG. 12 shows an extended dose matrix and isobologram demonstrating theeffects of combining Compound A3 and Compound B2 doses on proliferationof MCF7/ARO human breast carcinoma cells with Δ4A.

FIG. 13 shows an extended dose matrix and isobologram demonstrating theeffects of combining Compound A2 and Compound B2 doses on proliferationof MCF7/ARO human breast carcinoma cells with Δ4A.

FIG. 14 shows an extended dose matrix demonstrating the effects ofcombining Compound A3 and Compound B2 with or without the presence ofCompound C1 or Compound C3 on proliferation of MCF7/ARO human breastcarcinoma cells with Δ4A.

FIG. 15 shows an extended dose matrix demonstrating the effects ofcombining Compound A2 and Compound B2 with or without the presence ofCompound C1 or Compound C3 on proliferation of MCF7/ARO human breastcarcinoma cells with Δ4A.

FIG. 16 shows an extended dose matrix and isobologram demonstrating theeffects of combining Compound A3 and Compound B3 doses on proliferationof MCF7/ARO human breast carcinoma cells with Δ4A.

FIG. 17 shows an extended dose matrix and isobologram demonstrating theeffects of combining Compound A2 and Compound B3 doses on proliferationof MCF7/ARO human breast carcinoma cells with Δ4A.

FIG. 18 shows an extended dose matrix demonstrating the effects ofcombining Compound A3 and Compound B3 with or without the presence ofCompound C1 or Compound C3 on proliferation of MCF7/ARO human breastcarcinoma cells with Δ4A.

FIG. 19 shows an extended dose matrix demonstrating the effects ofcombining Compound A2 and Compound B3 with or without the presence ofCompound C1 or Compound C3 on proliferation of MCF7/ARO human breastcarcinoma cells with Δ4A.

FIGS. 20-22 show antitumor efficacy of various compounds used as singleagent, in double or in triple combination in the HBCx-34 human breastpatient-derived xenograft model.

FIG. 23 illustrates the study design of clinical trial described inExample 3.

FIGS. 24 and 25 show the duration of exposure to treatment in ARM1 andARM2 of the clinical trial described in Example 3 (interim results).

FIG. 26 shows the partial response observed for patient with metastaticbreast carcinoma treated with Compound A1 and Letrozole; the patientwith metastatic breast carcinoma had received a bilateralsalpingo-oophorectomy and right neck mass excision, and treatment withletrozole, fulvestrant, and the phosphatidylinositol 3-kinase inhibitorGDC0032 in the metastatic setting.

FIG. 27 illustrates the study design of clinical trial described inExample 5.

FIGS. 28 and 29 show the Mean plasma concentration-time profiles forCompound A1 and EVE in patients treated with Compound A1+EVE+EXE onC1D15.

FIG. 30 shows the duration of exposure to treatment of the clinicaltrial described in Example 5 (interim results).

FIG. 31 shows improvement in soft tissue metastases in a patient withlymph node, plura, lung, and soft tissue metastases who had received 1prior line of anastrozole and 1 prior line of fulvestrant in theadvanced/metastatic setting; the Cycle 3 Day 1 scan shows the largestarea of disease on follow-up.

DETAILED DESCRIPTION OF THE DISCLOSURE

The following general definitions are provided to better understand thedisclosure:

“Aromatase inhibitor” used herein relates to compounds which inhibit theestrogen production, i.e. the conversion of the substratesandrostenedione and testosterone to estrone and estradiol, respectively.Such compounds will be referred to as “aromatase inhibitors”.

“Selective estrogen receptor modulator (SERM)” refers to compound(s)that act on the estrogen receptor. A characteristic that distinguishesSERMs from pure receptor agonists and antagonists is that their actionis different in various tissues, thereby granting the possibility toselectively inhibit or stimulate estrogen-like action in varioustissues.

“PI3K inhibitor” is defined herein to refer to a compound which targets,decreases or inhibits phosphatidylinositol 3-kinase.Phosphatidylinositol 3-kinase activity has been shown to increase inresponse to a number of hormonal and growth factor stimuli, includinginsulin, platelet-derived growth factor, insulin-like growth factor,epidermal growth factor, colony-stimulating factor, and hepatocytegrowth factor, and has been implicated in processes related to cellulargrowth and transformation.

“Combination” refers to either a fixed combination in one dosage unitform, or a non-fixed combination (or kit of parts) for the combinedadministration where a compound and a combination partner (e.g. anotherdrug as explained below, also referred to as “therapeutic agent” or“co-agent”) may be administered independently at the same time orseparately within time intervals, especially where these time intervalsallow that the combination partners show a cooperative, e.g. synergisticeffect. The term “combined administration” or the like as utilizedherein are meant to encompass administration of the selected combinationpartner to a single subject in need thereof (e.g. a patient), and areintended to include treatment regimens in which the agents are notnecessarily administered by the same route of administration or at thesame time. The term “fixed combination” means that the activeingredients, e.g. a compound of formula A1 and a combination partner,are both administered to a patient simultaneously in the form of asingle entity or dosage. The terms “non-fixed combination” or “kit ofparts” mean that the active ingredients, e.g. a compound of formula A1and a combination partner, are both administered to a patient asseparate entities either simultaneously, concurrently or sequentiallywith no specific time limits, wherein such administration providestherapeutically effective levels of the two compounds in the body of thepatient.

“Treatment” includes prophylactic and therapeutic treatment (includingbut not limited to palliative, curing, symptom-alleviating,symptom-reducing) as well as the delay of progression of a cancerdisease or disorder. The term “prophylactic” means the prevention of theonset or recurrence of a cancer. The term “delay of progression” as usedherein means administration of the combination to patients being in apre-stage or in an early phase of the cancer to be treated, a pre-formof the corresponding cancer is diagnosed and/or in a patient diagnosedwith a condition under which it is likely that a corresponding cancerwill develop.

“Pharmaceutical preparation” or “pharmaceutical composition” refers to amixture or solution containing at least one therapeutic agent to beadministered to a warm-bloodeded, e.g., a human.

“Co-administer”, “co-administration” or “combined administration” or thelike are meant to encompass administration of the selected therapeuticagents to a single patient, and are intended to include treatmentregimens in which the agents are not necessarily administered by thesame route of administration or at the same time.

“Pharmaceutically acceptable” refers to those compounds, materials,compositions and/or dosage forms, which are, within the scope of soundmedical judgment, suitable for contact with the tissues of mammals,especially humans, without excessive toxicity, irritation, allergicresponse and other problem complications commensurate with a reasonablebenefit/risk ratio.

“Therapeutically effective” preferably relates to an amount of atherapeutic agent that is therapeutically or in a broader sense alsoprophylactically effective against the progression of a cancer.

“Jointly therapeutically effective” means that the therapeutic agentsmay be given separately (in a chronologically staggered manner,especially a sequence-specific manner) in such time intervals that theyprefer, in the warm-blooded animal, especially human, to be treated,still show a (preferably synergistic) interaction. Whether this is thecase can, inter alia, be determined by following the blood levels,showing that both compounds are present in the blood of the human to betreated at least during certain time intervals.

“Single pharmaceutical composition” refers to a single carrier orvehicle formulated to deliver effective amounts of both therapeuticagents to a patient. The single vehicle is designed to deliver aneffective amount of each of the agents, along with any pharmaceuticallyacceptable carriers or excipients. In some embodiments, the vehicle is atablet, capsule, pill, or a patch. In other embodiments, the vehicle isa solution or a suspension.

“Dose range” refers to an upper and a lower limit of an acceptablevariation of the amount of therapeutic agent specified. Typically, adose of the agent in any amount within the specified range can beadministered to patients undergoing treatment.

“Subject”, “patient”, or “warm-blooded animal” is intended to includeanimals. Examples of subjects include mammals, e.g., humans, dogs, cows,horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenicnon-human animals. In certain embodiments, the subject is a human, e.g.,a human suffering from, at risk of suffering from, or potentiallycapable of suffering from a brain tumor disease. Particularly preferred,the subject or warm-blooded animal is human.

The terms “about” or “approximately” usually means within 20%, morepreferably within 10%, and most preferably still within 5% of a givenvalue or range. Alternatively, especially in biological systems, theterm “about” means within about a log (i.e., an order of magnitude)preferably within a factor of two of a given value.

The present disclosure relates to a pharmaceutical combinationcomprising (1) a CDK inhibitor or a pharmaceutically acceptable saltthereof and (2) an anti-hormonal agent or a pharmaceutically acceptablesalt thereof.

The present disclosure also relates to a pharmaceutical combinationcomprising (1) a CDK inhibitor or a pharmaceutically acceptable saltthereof, (2) an anti-hormonal agent or a pharmaceutically acceptablesalt thereof, and (3) an agent that regulates the PI3K/Akt/mTOR pathwayor a pharmaceutically acceptable salt thereof.

Such combination may be for simultaneous, separate or sequential use forthe treatment of a cancer.

In one embodiment, the CDK inhibitor is CDK4/6 inhibitor.

The CDK4/6 inhibitor can be, for example,

Compound A1, described by Formula A1 below:

or,

Compound A2, described by Formula A2 below:

or,

palbociclib (hereinafter referred as Compound A3, also known asPD-0332991).

Compound A1 is also described by the chemical name7-Cyclopentyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid dimethylamide.

Compound A2 is also described by the chemical name7-cyclopentyl-N,N-dimethyl-2-(5-((1R,6S)-9-methyl-4-oxo-3,9-diazabicyclo[4.2.1]nonan-3-yl)pyridin-2-ylamino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide.

Compound A3 is also described by the chemical name6-Acetyl-8-cyclopentyl-5-methyl-2-{[5-(1-piperazinyl)-2-pyridinyl]amino}pyrido[2,3-d]pyrimidin-7(8H)-one.

In one embodiment, the anti-hormonal agent is an aromatase inhibitor.Such aromatase inhibitor can be either a non-steroidal aromataseinhibitor or a steroidal aromatase inhibitor.

Letrozole (hereinafter referred as Compound 1) is an example of anon-steroidal aromatase inhibitor.

Exemestane (hereinafter referred as Compound B2) is an example of asteroidal aromatase inhibitor.

In another embodiment, the anti-hormonal agent is an estrogen receptorantagonist.

Fulvestrant (hereinafter referred as Compound B3) is an example of anestrogen receptor antagonist.

In yet another embodiment, the anti-hormonal agent is a selectiveestrogen receptor modulator.

Tamoxifen (hereinafter referred as Compound B4) is an example of aselective estrogen receptor modulator.

In one embodiment, the agent that regulates the PI3K/Akt/mTOR pathway isa PI3K inhibitor.

The PI3K inhibitor can be, for example,

Compound C1, described by Formula C1 below:

or,

Compound C2, described by Formula C2 below:

Compound C1 is also described by the chemical name(S)-Pyrrolidine-1,2-dicarboxylic acid 2-amide1-({4-methyl-5-[2-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-pyridin-4-yl]-thiazol-2-yl}-amide).

Compound C2 is also described by the chemical name5-(2,6-di-4-morpholinyl-4-pyrimidinyl)-4-(trifluoromethyl)-2-pyrimidinamine.

In another embodiment, the agent that regulates the PI3K/Akt/mTORpathway is a mTOR inhibitor.

Everolimus (hereinafter referred as Compound C3) is an example of a mTORinhibitor.

Specific embodiments of the present disclosure include the following:

(1) Combination comprising Compound A1 and Compound E1;

(2) Combination comprising Compound A1 and Compound B2;

(3) Combination comprising Compound A1 and Compound B3;

(4) Combination comprising Compound A1 and Compound B4;

(5) Combination comprising Compound A2 and Compound E1;

(6) Combination comprising Compound A2 and Compound B2;

(7) Combination comprising Compound A2 and Compound B3;

(8) Combination comprising Compound A2 and Compound E4;

(9) Combination comprising Compound A3 and Compound E1;

(10) Combination comprising Compound A3 and Compound B2;

(11) Combination comprising Compound A3 and Compound B3;

(12) Combination comprising Compound A3 and Compound B4;

(13) Combination comprising Compound A1, Compound 1 and Compound C1;

(14) Combination comprising Compound A1, Compound 1 and Compound C2;

(15) Combination comprising Compound A1, Compound 1 and Compound C3;

(16) Combination comprising Compound A1, Compound B2 and Compound C1;

(17) Combination comprising Compound A1, Compound B2 and Compound C2;

(18) Combination comprising Compound A1, Compound B2 and Compound C3;

(19) Combination comprising Compound A1, Compound B3 and Compound C1;

(20) Combination comprising Compound A1, Compound B3 and Compound C2;

(21) Combination comprising Compound A1, Compound B3 and Compound C3;

(22) Combination comprising Compound A1, Compound B4 and Compound C1;

(23) Combination comprising Compound A1, Compound B4 and Compound C2;

(24) Combination comprising Compound A1, Compound B4 and Compound C3;

(25) Combination comprising Compound A2, Compound 1 and Compound C1;

(26) Combination comprising Compound A2, Compound 1 and Compound C2;

(27) Combination comprising Compound A2, Compound 1 and Compound C3;

(28) Combination comprising Compound A2, Compound B2 and Compound C1;

(29) Combination comprising Compound A2, Compound B2 and Compound C2;

(30) Combination comprising Compound A2, Compound B2 and Compound C3;

(31) Combination comprising Compound A2, Compound B3 and Compound C1;

(32) Combination comprising Compound A2, Compound B3 and Compound C2;

(33) Combination comprising Compound A2, Compound B3 and Compound C3;

(34) Combination comprising Compound A2, Compound B4 and Compound C1;

(35) Combination comprising Compound A2, Compound B4 and Compound C2;

(36) Combination comprising Compound A2, Compound B4 and Compound C3;

(37) Combination comprising Compound A3, Compound 1 and Compound C1;

(38) Combination comprising Compound A3, Compound 1 and Compound C2;

(39) Combination comprising Compound A3, Compound 1 and Compound C3;

(40) Combination comprising Compound A3, Compound B2 and Compound C1;

(41) Combination comprising Compound A3, Compound B2 and Compound C2;

(42) Combination comprising Compound A3, Compound B2 and Compound C3;

(43) Combination comprising Compound A3, Compound B3 and Compound C1;

(44) Combination comprising Compound A3, Compound B3 and Compound C2;

(45) Combination comprising Compound A3, Compound B3 and Compound C3;

(46) Combination comprising Compound A3, Compound B4 and Compound C1;

(47) Combination comprising Compound A3, Compound B4 and Compound C2;and

(48) Combination comprising Compound A3, Compound B4 and Compound C3.

The present disclosure further relates to the above pharmaceuticalcombination(s) for use in the treatment of a cancer.

The present disclosure further relates to a method for the treatment ofa cancer comprising administering the above pharmaceuticalcombination(s) in jointly therapeutically effective amount, to awarm-blooded animal, preferably a human, in need thereof.

In accordance with the present disclosure, the compounds in thepharmaceutical combination(s) may be administered either as a singlepharmaceutical composition, as separate compositions, or sequentially.

The present disclosure further relates to a kit comprising thepharmaceutical combination.

The Compounds A1-A3, B1-B4, C1-C3 may be incorporated in the combinationof the present disclosure in either the form of its free base or anysalt thereof. Salts can be present alone or in mixture with freecompound, e.g. the compound of the formula A1, and are preferablypharmaceutically acceptable salts. Such salts of the compounds offormula A1 are formed, for example, as acid addition salts, preferablywith organic or inorganic acids, from compounds of formula A1 with abasic nitrogen atom. Suitable inorganic acids are, for example, halogenacids, such as hydrochloric acid, sulfuric acid, or phosphoric acid.Suitable organic acids are, e.g., succinic acid, carboxylic acids orsulfonic acids, such as fumaric acid or methansulfonic acid. Forisolation or purification purposes it is also possible to usepharmaceutically unacceptable salts, for example picrates orperchlorates. For therapeutic use, only pharmaceutically acceptablesalts or free compounds are employed (where applicable in the form ofpharmaceutical preparations), and these are therefore preferred.

The Compounds A1-A3, B1-B4, C1-C3 can be synthesized by one skilled inthe art. Specifically, Compound A1 is disclosed as Example 74 ofWO2010/020675; Compound A2 is disclosed in WO2011/101409; Compound C1 isdisclosed as Example 15 of WO2010/029082; and Compound C2 is disclosedas Example 10 of WO2007/084786.

Suitable aromatase inhibitors include, but are not limited to,

-   -   (a) steroids, such as exemestane and formestane; and    -   (b) non-steroids, such as aminoglutethimide, vorozole,        fadrozole, anastrozole and, especially, letrozole.

Exemestane can be administered, e.g., in the form as it is marketed,e.g. under the trademark AROMASIN®. Formestane can be administered,e.g., in the form as it is marketed, e.g. under the trademark LENTARON®.Fadrozole can be administered, e.g., in the form as it is marketed, e.g.under the trademark AFEMA®. Anastrozole can be administered, e.g., inthe form as it is marketed, e.g. under the trademark ARIMIDEX®.Letrozole can be administered, e.g., in the form as it is marketed, e.g.under the trademark FEMARA® or FEMAR®. Letrozole has been specificallydescribed in the European patent No. 0 236 940 published on Sep. 16,1987, as well as in U.S. Pat. No. 4,978,672 published on Dec. 18, 1990,and Japanese Patent No. 2018112 all in the name of the applicant.Aminoglutethimide can be administered, e.g., in the form as it ismarketed, e.g. under the trademark ORIMETEN®.

The structure of the active agents identified by code nos., generic ortrade names may be taken from the actual edition of the standardcompendium “The Merck Index” or from databases, e.g., PatentsInternational (e.g., IMS World Publications). The corresponding contentthereof is hereby incorporated by reference.

Comprised are likewise the pharmaceutically acceptable salts thereof,the corresponding racemates, diastereoisomers, enantiomers, tautomers,as well as the corresponding crystal modifications of above disclosedcompounds where present, e.g. solvates, hydrates and polymorphs, whichare disclosed therein. The compounds used as active ingredients in thecombinations of the present disclosure can be prepared and administeredas described in the cited documents, respectively. Also within the scopeof this disclosure is the combination of more than two separate activeingredients as set forth above, i.e., a pharmaceutical combinationwithin the scope of this disclosure could include three activeingredients or more.

It is believed that the combination(s) of the present disclosurepossesses beneficial therapeutic properties, e.g. synergisticinteraction, strong in vitro or in vivo anti-proliferative activityand/or strong in vitro or in vivo antitumor response, which render itparticularly useful for the treatment of cancer.

Suitable cancers that can be treated with the combination of the presentdisclosure include, but are not limited to, sarcoma, lymphomas, cancerof the lung, bronchus, prostate, breast (including sporadic breastcancers and sufferers of Cowden disease), pancreas, gastrointestine,colon, rectum, colon, colorectal adenoma, thyroid, liver, intrahepaticbile duct, hepatocellular, adrenal gland, stomach, gastric, glioma,glioblastoma, endometrial, melanoma, kidney, renal pelvis, urinarybladder, uterine corpus, cervix, vagina, ovary, multiple myeloma,esophagus, a leukaemia, acute myelogenous leukemia, chronic myelogenousleukemia, lymphocytic leukemia, myeloid leukemia, brain, a carcinoma ofthe brain, oral cavity and pharynx, larynx, small intestine, non-Hodgkinlymphoma, melanoma, villous colon adenoma, a neoplasia, a neoplasia ofepithelial character, a mammary carcinoma, basal cell carcinoma,squamous cell carcinoma, actinic keratosis, tumor diseases (includingsolid tumors), a tumor of the neck or head, polycythemia vera, essentialthrombocythemia, myelofibrosis with myeloid metaplasia, and Waldenstroemdisease. Where a cancer, a tumor, a tumor disease, sarcoma, or a cancerare mentioned, also metastasis in the original organ or tissue and/or inany other location are implied alternatively or in addition, whateverthe location of the tumor and/or metastasis.

The combination of the present disclosure is particularly useful for thetreatment of a cancer mediated by phosphatidylinositol 3-kinase (PI3K),particularly the alpha-subunit of PI3K. Proliferative diseases mayinclude those showing overexpression or amplification of PI3K alpha,somatic mutation of PIK3CA or germline mutations or somatic mutation ofPTEN or mutations and translocation of p85a that serve to up-regulatethe p85-p110 complex. In a preferred embodiment, the cancer is a tumorand/or cancerous growth mediated by the alpha isoform of PI3K. Diseasemay include those showing overexpression or amplification of thealpha-isoform of PI3K and/or somatic mutation of PIK3CA.

The combination of the present disclosure is also particularly usefulfor the treatment of a hormone sensitive and/or hormone receptorpositive cancers. Hormone sensitive cancers may include, but are notlimited to, breast cancer, endometrial cancer, ovarian cancer, and/orcervical cancer. Hormone-receptor positive cancers may include estrogenreceptor positive cancers (i.e., cancer that grows in response to thehormone estrogen) or progesterone receptor positive cancers (ie., cancerthat grows in response to the hormone progesterone. Preferably, thehormone receptor positive cancer is estrogen receptor positive breastcancer.

In one embodiment, the cancer is a solid tumor.

In a further embodiment, the cancer is selected from the groupconsisting of cancer of the breast, endometrial, ovary and cervix.

In a further embodiment, the cancer is a cancer showing both (a)overexpression or amplification of the alpha-isoform of PI3K and/orsomatic mutation of PIK3CA, and (b) hormone receptor positive status.

In a further embodiment, the cancer is breast cancer. Preferably, thecancer is a breast cancer having either hormone receptor positive, amutation in the PIK3CA, or a combination thereof. More preferably, thecancer is estrogen receptor positive (+) breast cancer.

In a further embodiment, the cancer is a hormone receptor positive (+)breast cancer resistant to treatment with hormone therapy (e.g.,estrogen or progesterone). A cancer “resistant to treatment with hormonetherapy” refers to a cancer or tumor that either fails to respondfavorably to treatment with prior hormone therapy, or alternatively,recurs or relapses after responding favorably to hormone therapy. Saidhormone therapy is understood to be in the absence of a PI3K inhibitor.The cancer or tumor may be resistant or refractory at the beginning oftreatment or it may become resistant or refractory during treatment.

It is one objective of this disclosure to provide a pharmaceuticalcomposition comprising a quantity, which is jointly therapeuticallyeffective at targeting or preventing a cancer, of each therapeutic agentof the disclosure.

In accordance with the present disclosure, agents in the composition ofthe present disclosure may be administered together in a singlepharmaceutical composition, separately in two or more separate unitdosage forms, or sequentially. The unit dosage form may also be a fixedcombination.

The pharmaceutical compositions for separate administration of agents orfor the administration in a fixed combination (i.e., a single galenicalcomposition comprising at least two therapeutic agents according to thedisclosure may be prepared in a manner known per se and are thosesuitable for enteral, such as oral or rectal, topical, and parenteraladministration to subjects, including mammals (warm-blooded animals)such as humans, comprising a therapeutically effective amount of atleast one pharmacologically active combination partner alone, e.g., asindicated above, or in combination with one or more pharmaceuticallyacceptable carriers or diluents, especially suitable for enteral orparenteral application. Suitable pharmaceutical compositions contain,e.g., from about 0.1% to about 99.9%, preferably from about 1% to about60%, of the active ingredient(s).

Pharmaceutical compositions for the combination therapy for enteral orparenteral administration are, e.g., those in unit dosage forms, such assugar-coated tablets, tablets, capsules or suppositories, ampoules,injectable solutions or injectable suspensions. Topical administrationis e.g. to the skin or the eye, e.g. in the form of lotions, gels,ointments or creams, or in a nasal or a suppository form. If notindicated otherwise, these are prepared in a manner known per se, e.g.,by means of conventional mixing, granulating, sugar-coating, dissolvingor lyophilizing processes. It will be appreciated that the unit contentof each agent contained in an individual dose of each dosage form neednot in itself constitute an effective amount since the necessaryeffective amount can be reached by administration of a plurality ofdosage units.

Pharmaceutical compositions may comprise one or more pharmaceuticalacceptable carriers or diluents and may be manufactured in conventionalmanner by mixing one or both combination partners with apharmaceutically acceptable carrier or diluent. Examples ofpharmaceutically acceptable diluents include, but are not limited to,lactose, dextrose, mannitol, and/or glycerol, and/or lubricants and/orpolyethylene glycol. Examples of pharmaceutically acceptable acceptablebinders include, but are not limited to, magnesium aluminum silicate,starches, such as corn, wheat or rice starch, gelatin, methylcellulose,sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and, ifdesired, pharmaceutically acceptable disintegrators include, but are notlimited to, starches, agar, alginic acid or a salt thereof, such assodium alginate, and/or effervescent mixtures, or adsorbents, dyes,flavorings and sweeteners. It is also possible to use the compounds ofthe present disclosure in the form of parenterally administrablecompositions or in the form of infusion solutions. The pharmaceuticalcompositions may be sterilized and/or may comprise excipients, forexample preservatives, stabilizers, wetting compounds and/oremulsifiers, solubilisers, salts for regulating the osmotic pressureand/or buffers.

In particular, a therapeutically effective amount of each of thecombination partner of the combination of the disclosure may beadministered simultaneously or sequentially and in any order, and thecomponents may be administered separately or as a fixed combination. Forexample, the method of preventing or treating a cancer according to thedisclosure may comprise: (i) administration of the first agent in freeor pharmaceutically acceptable salt form; and (ii) administration of asecond agent in free or pharmaceutically acceptable salt form,simultaneously or sequentially in any order, in jointly therapeuticallyeffective amounts, preferably in synergistically effective amounts,e.g., in daily or intermittently dosages corresponding to the amountsdescribed herein. The individual combination partners of the combinationof the disclosure may be administered separately at different timesduring the course of therapy or concurrently in divided or singlecombination forms. Furthermore, the term administering also encompassesthe use of a pro-drug of a combination partner that convert in vivo tothe combination partner as such. The instant disclosure is therefore tobe understood as embracing all such regimens of simultaneous oralternating treatment and the term “administering” is to be interpretedaccordingly.

The effective dosage of each of combination partner agents employed inthe combination of the disclosure may vary depending on the particularcompound or pharmaceutical composition employed, the mode ofadministration, the condition being treated, the severity of thecondition being treated. Thus, the dosage regimen of the combination ofthe disclosure is selected in accordance with a variety of factorsincluding type, species, age, weight, sex and medical condition of thepatient; the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound employed. A physician, clinician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentration of drug withinthe range that yields efficacy requires a regimen based on the kineticsof the drug's availability to target sites. This involves aconsideration of the distribution, equilibrium, and elimination of adrug.

A further benefit is that lower doses of the active ingredients of thecombination of the disclosure can be used, e.g., that the dosages neednot only often be smaller but are also applied less frequently, or canbe used in order to diminish the incidence of side effects. This is inaccordance with the desires and requirements of the patients to betreated.

The combination of the agents can be combined in the same pharmaceuticalpreparation or in the form of combined preparations “kit of parts” inthe sense that the combination partners can be dosed independently or byuse of different fixed combinations with distinguished amounts of thecombination partners, i.e., simultaneously or at different time points.The parts of the kit of parts can then, e.g., be administeredsimultaneously or chronologically staggered, that is at different timepoints and with equal or different time intervals for any part of thekit of parts.

The present disclosure further relates to a kit comprising a firstcompound selected from the group consisting of Compounds A1-A3 orpharmaceutically acceptable salts thereof, a second compound selectedfrom the group consisting of Compounds B1-B4 or pharmaceuticallyacceptable salts thereof, and a package insert or other labelingincluding directions for treating a cancer.

The present disclosure further relates to a kit comprising a firstcompound selected from the group consisting of Compounds A1-A3 orpharmaceutically acceptable salts thereof, a second compound selectedfrom the group consisting of Compounds B1-B4 or pharmaceuticallyacceptable salts thereof, a third compound selected from the groupconsisting of Compounds C1-C3 or pharmaceutically acceptable saltsthereof, and a package insert or other labeling including directions fortreating a cancer.

The following Examples illustrate the disclosure described above; theyare not, however, intended to limit the scope of the disclosure in anyway. The beneficial effects of the pharmaceutical combination of thepresent disclosure can also be determined by other test models known assuch to the person skilled in the pertinent art.

Example 1

The following experimental procedure is performed to demonstrate theefficacy and anti-proliferative activity of Compound A1 in double ortriple combination in the treatment of breast cancer:

Preparation of Compounds/Reagent Solutions

Compound A1 (a CDK4/6 inhibitor, 10 mM), Compound 1 (Letrozole, Sigma,10 mM), Compound B3 (Fulvestrant, Sigma, 10 mM), Compound B2(Exemestane, Sigma, 10 mM), Compound C1 (a PI3K inhibitor, 10 mM),Compound C3 (an mTor inhibitor, 10 mM) and Compound C2 (a PI3Kinhibitor, 10 mM) were dissolved in DMSO. Δ4A (the precursorandrostenedione 10 mM) were dissolved in ethanol. All these reagentswere stored in aliquots at −20° C.

Cell Culture

MCF7 human breast carcinoma cells were provided by Dr. Chen Shiuan (Cityof Hope National Medical Center, CA, USA), which were stably transfectedwith the aromatase expression vector bearing the neomycin (G418)resistance gene (also named MCF7/Aro). Aromatase converts the precursorandrostenedione (Δ4A) into 17β-estradiol (E2), which is required for theproliferation of the host cell line. Unless otherwise mentioned, allcell culture reagents were obtained from Invitrogen. Cells weremaintained in MEM (#11095-080) supplemented with 10% v/v fetal bovineserum (FBS, #10099-141), 1 mM sodium pyruvate (#11360-070), 1% v/vnon-essential amino acids (#11140-050) and G418 (geneticin, #10131) in ahumidified incubator at 37° C. in 5% CO2. The cells were passaged twicea week and the medium was changed every 2 to 3 days. To assess estrogendriven cell proliferation, it was necessary to deplete the medium ofsteroids. To do so, the steroid-depleted (SD) medium, MEM (#51200-038,no phenol red & no glutamine) supplemented with charcoal stripped FBS(#12676-029) and Glutamax (#35050-061) was used. Medium without phenolred (pH indicator) was required since it is a structural homologue ofestrogen. Moreover, normal FBS need to be replaced by charcoal-strippedFBS in order to remove steroids. TryPLE Express (12604-013, no phenalred) was used for cell dissociation during SD treatment.

Cell Viability Assay and Cell Proliferation Assay

MCF7/Aro cells were steroid deprived for 3 days before trypsinized usingTryPLE Express (#12604-013, without phenol red) and 1500 cells/well wereplated on clear-bottom 384-well black plates (Greiner, #781091) intriplicates with 30 μl/well growth media, cells were allowed to attachovernight and were followed by 6 days of incubation with 10 nM of Δ4Aand various concentrations of drugs or drug combinations (10 μl/well).Cell viability was determined by measuring cellular ATP content usingthe CellTiter-Glo® (CTG) luminescent cell viability assay (Promega).Each single agent and combination treatment of cells was compared tocontrols (cells treated with an equivalent volume of medium). 30 ul/wellof the CTG reagents was added to each well at the end of the compoundtreatment and luminescence was recorded on an Envision plate reader(Perkin Elmer). Reduced and enhanced luminescent signal values(responses) were calculated relative to untreated (control) cells.

Combinations Tested

The following combinations were tested:

(a) Compound A1/Compound 1 (Letrozole);

(b) Compound A1/Compound 1 (Letrozole)/Compound C1 (1 uM or 500 nM);

(c) Compound A1/Compound 1 (Letrozole)/Compound C3 (20 nM or 2 nM);

(d) Compound A1/Compound 1 (Letrozole)/Compound C2 (333 nM);

(e) Compound A1/Compound B3 (Fulvestrant);

(f) Compound A1/Compound B3 (Fulvestrant)/Compound C1 (1 uM or 500 nM);

(g) Compound A1/Compound B3 (Fulvestrant)/Compound C3 (20 nM or 2 nM);

(h) Compound A1/Compound B3 (Fulvestrant)/Compound C2 (333 nM);

(i) Compound A1/Compound B2 (Exemestane);

(j) Compound A1/Compound B2 (Exemestane)/Compound C1 (1 uM or 500 nM);

(k) Compound A1/Compound B2 (Exemestane)/Compound C3 (20 nM or 2 nM);and

(1) Compound A1/Compound B2 (Exemestane)/Compound C2 (333 nM).

Compound A1, Compoud 1 (Letrozole), Compoud B2 (Exemestane) and CompoudB3 (Fulvestant) were in multiple doses, and Compoud C1, Compoud C2 andCompoud C3 were in a single dose as a background compounds (doses aslabeled above) in all the triple combinations.

To evaluate the anti-proliferative activity of all the combinations in anon-bias way, as well as to identify synergistic effect at all possibleconcentrations, the studies were conducted with a “dose matrix.” Thisutilized all possible permutations of serially-diluted CompoundA1/Compound 1 (Letrozole), Compound A1/Compound B3 (Fulvestant) andCompound A1/Compound B2 (Exemestane) (with a single dose backgroundcompound). In all combination assays, agents were appliedsimultaneously.

The “dose matrix, Compound A1/Compound 1, Compound A1/Compound B3 andCompound A1/Compound B2” were consisted of the followings:

-   -   (a) Compound A1 (in the combination of Compound A1/Compound 1        and Compound A1/Compound B2), which was subjected to a 6 dose 3×        serial dilution with a high dose of 10 uM and a low dose of        approximately 41 nM    -   (b) Compound A1 (in the combination of Compound A1/Compound B3),        which was subjected to a 5 or 6 dose 3× serial dilution with a        high dose of 1 or 3 uM and a low dose of approximately 12 nM    -   (c) Compound 1, which was subjected to a 7 dose 3× serial        dilution with a high dose of 5 uM and a low dose of        approximately 7 nM    -   (d) Compound B3, which was subjected to a 6 dose 3× serial        dilution with a high dose of 800 nM and a low dose of        approximately 3 nM    -   (e) Compound B2, which was subjected to a 7 dose 3× serial        dilution with a high dose of 10 uM and a low dose of        approximately 14 nM.

Calculating the Effect of Combinations:

The synergistic interaction (analyzed using Chalice software[CombinatoRx, Cambridge Mass.]) was calculated by comparing the responsefrom a combination to the response of the agent acting alone, againstthe drug-with-itself dose-additive reference model. Deviations from doseadditives can be assessed numerically with a Combination Index (CI),which quantifies the overall strength of combination effect. Thiscalculation (essentially a volume score) is as follows:V_(HSA)=Σ_(X,Y)lnfX lnfY (I_(data)−I_(HSA)). Additionally, CI iscalculated between the data and the highest single-agent surface,normalized for single agent dilution factors (Lehar et al, 2009):

Data Analysis

Data evaluation and graph generation were performed using MicrosoftExcel software, and Chalice software.

Results

To investigate the activity of double or triple combinations of CompoundA1 with antiestrogen therapeutics such as fulvestrant (Compound B3),letrozole (Compound 1) and exemestane (Compound B2), with or withoutPI3K or mTOR inhibitor Compound C1, Compound C2 or Compound C3 on cellproliferation, various combos as described in the method section weretested in androstenedione driven, aromatase overexpressing MCF7 cells.Synergy was observed between Compound A1 and all three antihormonaltherapies in a 7×8 dose matrix combination setting, with synergy scoreeach at 4.12, 2.41 and 1.43, for letrozole (Compound 1), exemestane(Compound B2) and fulvestrant (Compound B3), respectively. Various doesof PI3K and mTOR inhibitor was also added to the same 7×8 does matrixsetting as a background compounds to test the efficacy of triplecombinations, in all cases, the triple combo significantly enhanced themaximum level of inhibition achieved by single or double reagents, andgreatly reduced the doses needed for achieving the same levels ofinhibition. Those results solidly support the concept of combining twoor three reagents targeting cell cycle, mTOR/PI3K and estrogen pathwayin ER positive breast cancer.

The results from Example 1 are shown in FIGS. 1-7.

Example 2

The following experimental procedure is performed to demonstrate theefficacy and anti-proliferative activity of Compound A2 or Compound A3in double or triple combination in the treatment of breast cancer:

Preparation of Compounds/Reagent Solutions

Compound A2 (a CDK4/6 inhibitor, 10 mM), Compound A3 (a CDK4/6inhibitor, 10 mM), Compound 1 (Letrozole, Sigma, 10 mM), Compound B3(Fulvestrant, Sigma, 10 mM), Compound B2 (Exemestane, Sigma, 10 mM),Compound C1 (a PI3K inhibitor, 10 mM), and Compound C3 (an mTorinhibitor, 10 mM) were dissolved in DMSO. Δ4A (the precursorandrostenedione, 10 mM) were dissolved in ethanol. All these reagentswere stored in aliquots at −20° C.

Cell Culture

MCF7 human breast carcinoma cells were provided by Dr. Chen Shiuan (Cityof Hope National Medical Center, CA, USA), which were stably transfectedwith the aromatase expression vector bearing the neomycin (G418)resistance gene (also named MCF7/Aro). Aromatase converts the precursorandrostenedione (Δ4A) into 17β-estradiol (E2), which is required for theproliferation of the host cell line. Unless otherwise mentioned, allcell culture reagents were obtained from Invitrogen. Cells weremaintained in MEM (#11095-080) supplemented with 10% v/v fetal bovineserum (FBS, #10099-141), 1 mM sodium pyruvate (#11360-070), 1% v/vnon-essential amino acids (#11140-050) and G418 (geneticin, #10131) in ahumidified incubator at 37° C. in 5% CO2. The cells were passaged twicea week and the medium was changed every 2 to 3 days. To assess estrogendriven cell proliferation, it was necessary to deplete the medium ofsteroids. To do so, the steroid-depleted (SD) medium, MEM (#51200-038,no phenol red & no glutamine) supplemented with charcoal stripped FBS(#12676-029) and Glutamax (#35050-061) was used. Medium without phenolred (pH indicator) was required since it is a structural homologue ofestrogen. Moreover, normal FBS need to be replaced by charcoal-strippedFBS in order to remove steroids. TryPLE Express (12604-013, no phenalred) was used for cell dissociation during SD treatment.

Cell Viability Assay and Cell Proliferation Assay

MCF7/Aro cells were steroid deprived for 3 days before trypsinized usingTryPLE Express (#12604-013, without phenol red) and 1500 cells/well wereplated on clear-bottom 384-well black plates (Greiner, #781091) intriplicates with 30 μl/well growth media, cells were allowed to attachovernight and were followed by 6 days of incubation with 10 nM of Δ4Aand various concentrations of drugs or drug combinations (10 μl/well).Cell viability was determined by measuring cellular ATP content usingthe CellTiter-Glo® (CTG) luminescent cell viability assay (Promega).Each single agent and combination treatment of cells was compared tocontrols (cells treated with an equivalent volume of medium). 30 ul/wellof the CTG reagents was added to each well at the end of the compoundtreatment and luminescence was recorded on an Envision plate reader(Perkin Elmer). Reduced and enhanced luminescent signal values(responses) were calculated relative to untreated (control) cells.

Combinations Tested

The following combinations were tested:

(a) Compound A2/Compound 1 (Letrozole);

(b) Compound A2/Compound 1 (Letrozole)/Compound C1;

(c) Compound A2/Compound 1 (Letrozole)/Compound C3;

(d) Compound A2/Compound B3 (Fulvestrant);

(e) Compound A2/Compound B3 (Fulvestrant)/Compound C1;

(f) Compound A2/Compound B3 (Fulvestrant)/Compound C3;

(g) Compound A2/Compound B3 (Exemestane);

(h) Compound A2/Compound B3 (Exemestane)/Compound C1;

(i) Compound A2/Compound B3 (Exemestane)/Compound C3;

(j) Compound A3/Compound 1 (Letrozole);

(k) Compound A3/Compound 1 (Letrozole)/Compound C1;

(l) Compound A3/Compound 1 (Letrozole)/Compound C3;

(m) Compound A3/Compound B3 (Fulvestrant);

(n) Compound A3/Compound B3 (Fulvestrant)/Compound C1;

(o) Compound A3/Compound B3 (Fulvestrant)/Compound C3;

(p) Compound A3/Compound B3 (Exemestane);

(q) Compound A3/Compound B3 (Exemestane)/Compound C1; and

(r) Compound A3/Compound B3 (Exemestane)/Compound C3.

Compound A2, Compound A3, Letrozole (Compound 1), Exemestane (CompoundE2) and Fulvestant (Compound E3) were in multiple doses, and Compound C1(1 uM) and Compound C2 (20 nM) were in a single dose as a background inall the triple combinations.

To evaluate the anti-proliferative activity of all the combinations in anon-bias way, as well as to identify synergistic effect at all possibleconcentrations, the studies were conducted with a “dose matrix.” Thisutilized all possible permutations of serially-diluted CompoundA2/Compound 1 (Letrozole), Compound A2/Compound B3 (Fulvestant),Compound A2/Compound B2 (Exemestane), Compound A3/Compound 1(Letrozole), Compound A3/Compound B3 (Fulvestant) and CompoundA3/Compound B2 (Exemestane) (with a single dose background compound). Inall combination assays, agents were applied simultaneously.

The “dose matrix, Compound A2/Compound 1, Compound A2/Compound B3,Compound A2/Compound B2, Compound A3/Compound 1, Compound A3/Compound B3and Compound A3/Compound E2” were consisted of the followings:

-   -   (a) Compound A2, which was subjected to a 7 dose 3× serial        dilution with a high dose of 3 uM and a low dose of        approximately 4.1 nM    -   (b) Compound A3, which was subjected to a 7 dose 3× serial        dilution with a high dose of 3 uM and a low dose of        approximately 4.1 nM    -   (c) Compound 1, which was subjected to a 6 dose 3× serial        dilution with a high dose of 5 uM and a low dose of        approximately 20.6 nM    -   (d) Compound B3, which was subjected to a 6 dose 3× serial        dilution with a high dose of 100 nM and a low dose of        approximately 0.4 nM    -   (e) Compound B2, which was subjected to a 6 dose 3× serial        dilution with a high dose of 10 uM and a low dose of        approximately 41.2 nM

Calculating the Effect of Combinations

The synergistic interaction (analyzed using Chalice software[CombinatoRx, Cambridge Mass.]) was calculated by comparing the responsefrom a combination to the response of the agent acting alone, againstthe drug-with-itself dose-additive reference model. Deviations from doseadditives can be assessed numerically with a Combination Index (CI),which quantifies the overall strength of combination effect. Thiscalculation (essentially a volume score) is as follows:V_(HSA)=Σ_(X,Y)lnfX lnfY (I_(data)−I_(HSA)). Additionally, CI iscalculated between the data and the highest single-agent surface,normalized for single agent dilution factors (Lehar et al, 2009):

Data Analysis

Data evaluation and graph generation were performed using MicrosoftExcel software, and Chalice software.

Results

To investigate the activity of double or triple combinations of CompoundA2 and Compound A3 with antiestrogen therapeutics such as fulvestrant(Compound B3), letrozole (Compound 1) and exemestane (Compound B2), withor without PI3K or mTOR inhibitor Compound C1 or Compound C3 on cellproliferation, various combos as described in the method section weretested in androstenedione driven, aromatase overexpressing MCF7 cells.Synergy was observed between Compound A3 and all three antihormonaltherapies in a 7×8 dose matrix combination setting, with score each at3.7, 1.2 and 1.7 for letrozole, exemestane and fulvestrant respectively.And synergy was also observed in Compound A2/Letrozole and CompoundA2/Fulvestrant combinations with score each at 3.2 and 1.4. Single doseof PI3K and mTOR inhibitor was also added to the same 7×8 does matrixsetting as a background compounds to test the efficacy of triplecombinations, in all cases, the triple combo significantly enhanced themaximum level of inhibition achieved by single or double reagents, andgreatly reduced the doses needed for achieving the same levels ofinhibition. Those results solidly support the concept of combining twoor three reagents targeting cell cycle, mTOR/PI3K and estrogen pathwayin ER positive breast cancer.

The results from Example 2 are shown in FIGS. 8-19.

The Table below summerizes the synergy score of the various combinationstested in Example 2.

Combo synergy score Compound A3/Compound B1 3.7 Compound A3/CompoundB1/Compound C1 1.7 Compound A3/Compound B1/Compound C3 4.5 CompoundA3/Compound B2 1.2 Compound A3/Compound B2/Compound C1 1.5 CompoundA3/Compound B2/Compound C3 2.8 Compound A3/Compound B3 1.7 CompoundA3/Compound B3/Compound C1 3.0 Compound A3/Compound B3/Compound C3 1.7Compound A2/Compound B1 3.2 Compound A2/Compound B1/Compound C1 1.9Compound A2/Compound B1/Compound C3 4.4 Compound A2/Compound B2 0.8Compound A2/Compound B2/Compound C1 1.3 Compound A2/Compound B2/CompoundC3 2.4 Compound A2/Compound B3 1.4 Compound A2/Compound B3/Compound C13.2 Compound A2/Compound B3/Compound C3 1.5

Example 3

A clinical trial is currently on going to further the clinicaldevelopment of the two investigational agents in ER+ breast cancer,Compound A1 (CDK4/6 inhibitor) and Compound C1 (PI3K inhibitor). This isa multi-center, open-label, dose finding Phase Ib/II trial. The Phase Ibpart is a three-part dose escalation study to estimate the MTD and/orRP2D for two double combinations: Compound A1 with letrozole andCompound C1 with letrozole followed by estimation of the MTD and/or RP2Dof the triple combination of Compound A1+Compound C1 with letrozole.

The three-part Phase Ib will be followed by a randomized Phase II studyto assess the preliminary anti-tumor activity of the two doublecombination regimens (Compound A1+letrozole and Compound C1+letrozole)versus the triple combination (Compound A1+Compound C1 with letrozole)and to further evaluate their safety in patients with ER+/HER2− locallyadvanced or metastatic breast cancer.

Approximately 290 adult women with ER+/HER2− locally advanced ormetastatic breast cancer will be enrolled.

The starting dose for the study drug combination doublets and tripletare described below. The standard dose of letrozole will be usedthroughout this study (2.5 mg/day).

Starting Doses for Each Arm:

Compound A1 (3 weeks followed by a one week Compound C1 Letrozole Armbreak) (QD) (QD) Compound A1 and 600 mg — 2.5 mg Letrozole Compound C1and — 300 mg 2.5 mg Letrozole Compound A1 and 400 mg 100 mg 2.5 mgCompound C1 and LetrozoleThe objectives of the Phase Ib portion of the study are:

Primary Objectives

-   -   To estimate the maximum tolerated dose (MTD) and/or recommended        Phase II dose (RP2D) of the following combinations:    -   Arm 1: Compound A1+letrozole (2.5 mg)    -   Arm 2: Compound C1+letrozole (2.5 mg)    -   Arm 3: Compound A1+Compound C1+letrozole (2.5 mg).

Secondary Objectives

-   -   To characterize the pharmacokinetic (PK) profiles of Compound        A1, Compound C1, and letrozole when used in combination.    -   To characterize the safety and tolerability in Arms 1, 2, and 3.    -   To assess preliminary clinical antitumor activity in Arms 1, 2,        and 3.

Study Design (FIG. 23)

-   -   In the Phase Ib portion of this multicenter, open-label study,        postmenopausal women with ER+/human epidermal growth factor        receptor negative (HER2-) advanced BC are being treated with        once-daily doses of Compound A1        (3-weeks-on/1-week-off)+letrozole (2.5 mg) or Compound        C1+letrozole (2.5 mg).    -   Dose escalation is guided by the adaptive Bayesian Logistic        Regression Model (BLRM) along with the Escalation With Overdose        Control principle.    -   PK assessments were conducted prior to dose-escalation decisions        during the study to monitor exposure and evaluate possibility of        cytochrome P450-mediated drug-drug interactions.    -   Upon determination of the MTD/RP2D in Arms 1 and 2, the BLRM        will be updated with the most recent data from the        dose-escalation in Arms 1 and 2, and this will be used to        determine the starting dose for Arm 3.

Key Inclusion Criteria

-   -   Postmenopausal women with metastatic or locally advanced        ER+/HER2− BC.    -   Any number of prior lines of endocrine therapy.    -   Up to 1 prior cytotoxic regimen in the metastatic or locally        advanced setting.    -   Representative tumor specimen (archival or new) available for        molecular testing (unless otherwise agreed).    -   Newly obtained, matched pre- and on-therapy tumor samples are        mandatory in the Phase Ib dose-escalation part of the study.

Key Exclusion Criteria

-   -   Prior treatment with a CDK4/6, AKT, mTOR, or PI3K inhibitor and        failure to benefit.    -   Current symptomatic brain metastases.    -   Clinically manifest diabetes mellitus, history of gestational        diabetes mellitus, or documented steroid-induced diabetes        mellitus.    -   QT corrected with Fridericia's formula (QTcF)>470 ms.

Assessments

-   -   Routine safety assessments conducted at baseline and at regular        intervals throughout the study, and adverse events (AEs)        assessed continuously according to Common Terminology Criteria        for Adverse Events v4.03.    -   Tumor response evaluated locally by the investigator, using        computerized tomography and magnetic resonance imaging, based on        Response Evaluation Criteria In Solid Tumors v1.1. Evaluations        conducted at baseline, every 8 weeks through to Cycle 6, every        12 weeks thereafter (or sooner if there is clinical evidence of        disease progression), and at end of treatment.    -   Samples for PK evaluations collected on Days 1, 2, 8, 15, 21,        and 22 of Cycle 1 and on Day 15 of Cycles 2-6. Real-time PK        assessments were conducted to guide dose escalation (in addition        to BLRM).

Interim Results Patient Characteristics and Disposition

-   -   10 patients have been treated with Compound A1 and letrozole        (Arm 1), and 7 patients have been treated with Compound C1 and        letrozole (Arm 2). The patients details are shown in Table 1.

TABLE 1 Patient Characteristics and Disposition Arm 1: Compound Arm 2:Compound A1 600 mg + C1 300 mg + All subjects Characteristic letrozole(n = 10) letrozole (n = 7) (N = 17) Median age, years (range) 59 (45-67)61 (51-72) 60 (45-72) WHO performance status, n (%)  0 5 (50) 4 (57) 9(53)  1 5 (50) 3 (43) 8 (47) Median time since initial 123 (9-173) 49(2-295) 104 (2-295) diagnosis to first dose of treatment, months (range)Pts who received prior 10 (100) 7 (100) 17 (100) antineoplasticregimens, n (%) Number of regimens, n (%)  1 2 (20) 3 (43) 5 (29)  2 1(10) 2 (29) 3 (18)  4 0 1 (14) 1 (6)  5 2 (20) 0 2 (12) >5 5 (50) 1 (14)6 (35) Prior therapies received in the advanced/metastatic setting, n(%) Chemotherapy 4 (40) 0 4 (24) Anastrozole 4 (40) 3 (43) 7 (41)Fulvestrant 6 (60) 2 (29) 8 (47) Letrozole 4 (40) 3 (43) 7 (41)Tamoxifen 1 (10) 0 1 (6) Exemestane 3 (30) 2 (29) 5 (29) PI3K/AKT/mTOR 5(50) 1 (14) 6 (35) inhibitors Other 7 (70) 2 (29) 9 (53) Number of ptswho received 9 (90) 7 (100) 16 (94) prior surgery, n (%) Number of ptswho received 8 (80) 4 (57) 12 (71) prior radiotherapy, n (%) mTOR,mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; pts,patients; WHO, World Health Organization.

-   -   At the time of study entry, all patients had stage IV ER+/HER2−        BC.    -   Treatment has been discontinued in 2 (20%) patients in Arm 1 due        to disease progression. At the cut-off date, treatment was        ongoing for all 7 (100%) patients in Arm 2.

Safety

-   -   Of 12 patients evaluable as part of the dose-determining set (6        in each arm), 3 dose-limiting toxicities (DLTs) were observed: 1        Grade 4 neutropenia in Arm 1 and 2 Grade 2 hyperglycemia in Arm        2.    -   The most common (>30% patients) all-grade adverse events        suspected to be study drug-related were (see Table 2):    -   Arm 1: neutropenia (90%) and nausea (40%)    -   Arm 2: hyperglycemia (57%), nausea (43%), decreased appetite        (43%), and diarrhea (43%).

TABLE 2 All Grades ≥10% and All Grade ¾ Adverse Events Arm 1 Compound A1Arm 2: Compound C1 600 mg + letrozole 300 mg + letrozole All subjects (n= 10) (n = 7) (N = 17) All Grade All Grade All Grade Adverse grades, ¾,grades, ¾, grades, ¾, event n (%) n (%) n (%) n (%) n (%) n (%)Hematologic adverse events Neutropenia 9 (90) 5 (50) 0 0 9 (53)  5 (29)Leukopenia 2 (20) 0 0 0 2 (12) 0 Lymphopenia 0 0 1 (14) 1 (14) 1 (6)  1(6) Non-hematologic adverse events Nausea 4 (40) 0 3 (43) 0 7 (41) 0Fatigue 3 (30) 0 2 (29) 1 (14) 5 (29) 1 (6) Decreased 1 (10) 0 3 (43) 04 (24) 0 appetite Diarrhea 1 (10) 0 3 (43) 0 4 (24) 0 Hyperglycemia 0 04 (57) 1 (14) 4 (24) 1 (6) Weight 1 (10) 0 2 (29) 0 3 (18) 0 decreasedDysgeusia 0 0 2 (29) 0 2 (12) 0

-   -   QTcF prolongation (>470 ms) was not observed in Arm 1.    -   Grade 3/4 adverse events suspected to be study drug related        included (Table 2):        -   Arm 1: neutropenia (50%)        -   Arm 2: lymphopenia (14%), fatigue (14%), and hyperglycemia            (14%).    -   Dose reductions occurred in 5 patients: 1 patient in Arm 1 and 4        patients in Arm 2.

Pharmacokinetics

-   -   Preliminary PK data for Compound A1, Compound C1, and letrozole        are as follows (Table 3):        -   PK for Compound A1 and Compound C1 on Days 1 and 21 are            comparable with historic single-agent data.        -   PK for letrozole on Day 1 are comparable with those observed            in single-agent studies.        -   Additional data are being gathered from patients currently            enrolled in the trial to further evaluate letrozole PK in            combination with Compound A1.

TABLE 3 Pharmacokinetic Parameters of Compound A1, Compound C1 andLetrozole AUC all C_(max) (h*ng/ml), (ng/ml), T_(max) (h), Analyte nmean (SD) mean (SD) median (range) Arm 1, C1D1 Compound A1 6 13072(9458) 1320 (859) 3.0 (1.9-4.2) Letrozole 6 421 (115) 25 (4) 2.0 Arm 1,C1D21 Compound A1 3 32038 (16586) 2780 (767) 4.0 (1.9-4.0) Letrozole 31364 (785) 79 (12) 4.0 (4.0-7.5) Arm 2, C1D1 Compound C1 5 29102 (8251)2480 (922) 3.8 (1.2-4.1) Letrozole 5 330 (115) 27 (13) 2.0 (1.2-4.0) Arm2, C1D21 Compound C1 3 40358 (6329) 3167 (314) 2.0 (2.0-3.8) Letrozole 32412 (189) 118 (9) 2.0 (1.9-2.0) AUC, area under the curve; C, cycle;Cmax, maximum concentration; D, day; Tmax, time to reach maximumconcentration.

Clinical Activity

-   -   Duration of exposure to treatment is shown in FIGS. 24 and 25.    -   In Arm 1, there was 1 patient with a confirmed partial response        (FIG. 26), 2 patients with stable disease (SD), and 1 patient        without measurable disease had neither complete response nor        progressive disease (NCRNPD; FIG. 24).    -   In Arm 2, there were 2 patients with SD and 3 patients had        NCRNPD (FIG. 25).

Conclusion (Based on Interim Results)

Both arms of the study have demonstrated an acceptable safety profileand preliminary signs of clinical activity in postmenopausal women withER+/HER2− advanced BC.

-   -   Neutropenia is an anticipated side effect of Compound A1,        potentially due to inhibition of proliferation via CDK4/6        inhibition.    -   Hyperglycemia observed in Arm 2 (Compound C1+letrozole) may be        an on-target effect of PI3K inhibition.    -   Dose escalation continues to determine the MTD/RP2D.    -   Upon determination of the MTD/RP2D in Arms 1 and 2, enrollment        into Arm 3 will commence. Following the Phase Ib portion of the        study a randomized Phase II portion will compare Compound        A1+letrozole and Compound C1+letrozole with Compound A1+Compound        C1+letrozole.

Example 4

A multi-center, pre-surgical, randomized, phase II study is planned, toassess the biological activity of Compound A1, 400 mg or 600 mg daily,in combination with letrozole 2.5 mg daily, as compared to single agentletrozole daily in postmenopausal patients with newly diagnosed HR+,HER2-negative, early breast cancer. A total of approximately 120patients will be randomized. Patients will receive trial therapy for 14days (±3 days) and then undergo surgery. Patients will be randomlyassigned to treatment with:

-   -   a. Letrozole (2.5 mg once daily); OR    -   b. Letrozole (2.5 mg once daily)+Compound A1 400 mg daily; OR    -   c. Letrozole (2.5 mg once daily)+Compound A1 600 mg daily

The primary objective of the study is to assess the cell cycle responserate defined as the percentage of patients who achieve a reduction inKi67 expression to natural logarithm of percentage positive Ki67 of lessthan 1 (Baselga 2009). Although the trial is designed as open label, allpharmacodynamics and clinical pharmacology endpoints will be assessed byexperts who are blinded to randomized treatment.

Example 5

A Phase Ib/II Trial of Compound A1 with everolimus and exemestane in thetreatment of ER+ Her2− Advanced Breast Cancer is on-going. The purposeof the trial is to estimate the MTD(s) and/or RP2D of Compound A1 incombination with everolimus+exemestane, and Compound A1 in combinationwith exemestane, and to characterize the safety and tolerability of thecombinations of everolimus+exemestane±Compound A1 and CompoundA1+exemestane in patients with ER+ HER2− advanced breast cancer. Thestudy consists of 3 arms:

Arms Assigned Interventions Compound A1 + everolimus + Compound A1 istaken orally once per exemestane triple combination day for 21 days ofeach 28 day cycle. Exemestane is taken orally once per day. Everolimusis taken orally once per day. Compound A1 + exemestane Compound A1 istaken orally once per double combination day for 21 days of each 28 daycycle Exemestane is taken orally once per day. everolimus + exemestaneExemestane is taken orally once per day. double combination Everolimusis taken orally once per day.Compound A1 comes in 50 mg and 200 mg capsules. Exemestane comes in 25mg tablets. Everolimus comes in 2.5 mg, 5 mg, and 7.5 mg tablets.The objectives of the Phase Ib portion of this study are:

Primary Objective

Determine the maximum tolerated dose (MTD)/recommended Phase II dose(RP2D) of Compound A1+everolimus (EVE)+exemestane (EXE) in patients withER+/human epidermal growth factor receptor 2-negative (HER2-) advancedBC.

Secondary Objectives

Determine the safety and tolerability of Compound A1+EVE+EXE andCompound A1+EXE.

Characterize the pharmacokinetics (PK) of Compound A1 and/or EVE whenadministered in combination with EXE.

Assess preliminary antitumor activity of Compound A1+EVE+EXE andCompound A1+EXE.

Evaluate the relationship between antitumor activity and molecularaberrations in the cyclin D-CDK4/6-INK-Rb, PI3K/AKT/mTOR, and othercancer-related pathways.

Study Design:

In the Phase Ib portion of this Phase Ib/II multicenter, open-labelstudy, postmenopausal women with ER+/HER2− advanced BC, resistant toletrozole or anastrozole, are being treated with escalating doses ofCompound A1+EVE+EXE (25 mg/day) or a safety run-in of Compound A1 (600mg/day)+EXE (25 mg/day; FIG. 27).

Dose escalation is being guided by the adaptive Bayesian LogisticRegression Model along with the Escalation with Overdose Controlprinciple and PK was assessed prior to dose-escalation decisions.

Upon determination of the MTD/RP2D, the Phase II portion of the studywill compare Compound A1+EVE+EXE triplet) and Compound A1+EXE (doublet)with EVE+EXE.

Key Inclusion Criteria:

Postmenopausal women with ER+/HER2− locally advanced or metastatic BC.

Recurrence while on, or within 12 months of end of, adjuvant treatmentwith letrozole or anastrozole OR progression while on, or within 1 monthof end of, treatment with letrozole or anastrozole treatment for locallyadvanced or metastatic BC. Letrozole or anastrozole need not be the lasttreatment prior to study start.

Previous treatment with a CDK4/6 inhibitor, EXE, or mTOR inhibitorallowed (for Phase Ib but not Phase II).

Representative tumor specimen (archival or new) available for moleculartesting.

Key Exclusion Criteria:

>2 chemotherapy lines for advanced BC.

Absolute neutrophil count ≤1.5×109/L.

QT corrected with Fridericia's formula >470 ms.

Assessments:

Routine safety assessments conducted at baseline and at regularintervals throughout the study. Adverse events (AEs) are being assessedcontinuously according to Common Terminology Criteria for Adverse Eventsv4.03.

Tumor response assessed locally by the investigator using computerizedtomography or magnetic resonance imaging according to ResponseEvaluation Criteria In Solid Tumors v1.1 at baseline and on Day (D) 1 ofCycles (C) 3, 5, and 7, on D1 of every 4th subsequent cycle (or soonerif clinically indicated), and at the end of treatment.

PK evaluations for Compound A1 and EVE performed in patients treatedwith Compound A1+EVE+EXE during C1 on D1, 2, 8, 15, 16, and 21, and D1of each subsequent cycle up to and including C6.

Tumor samples analyzed by next-generation sequencing to determine anyalterations in genes of interest.

Interim Results: Patient Characteristics and Disposition:

As of the interim report cut of date, 16 patients have been treated: 3patients with Compound A1 600 mg+EXE 25 mg and 13 patients with CompoundA1 (200 mg [6 patients]; 300 mg [6 patients]; 250 mg [1 patient])+EVE2.5 mg+EXE 25 mg.

Treatment has been discontinued in 5 (31%) patients. The primary reasonsfor discontinuation were: disease progression (4 patients) and death (1patient).

In the advanced/metastatic setting, previous treatment with letrozole oranastrozole was reported in 10 (63%) and 5 (31%) patients, respectively,while 6 (38%) and 3 (19%) patients had received prior EXE and EVE,respectively (Table 4).

TABLE 4 Patient and Disease Characteristics Characteristic All (N = 16)Median age, years (range) 57 (41-84) 57 (41-84) Time since initialdiagnosis of primary site 83 (8-355) to first dose of drug (months),median (range) Site of metastases, n (%) Bone (no visceral disease) 3(25) Bone and visceral 9 (50) Visceral (no bone disease) 4 (25) Others 9(56) Setting at last medication, n (%) Adjuvant 2 (13) Neoadjuvant 1 (6)Advanced/metastatic disease 14 (88) Number of prior regimens in theadvanced/metastatic setting, n (%)  0 2 (13) 1-2 5 (31) 3-4 7 (44) >4 2(13) Number of prior chemotherapy regimens in the advanced/metastaticsetting, n (%)  0 10 (63)  1 2 (13)  2 4 (25) Prior therapies receivedin the advanced/metastatic setting, n (%) Letrozole 10 (63) Anastrozole5 (31) Fulvestrant 11 (69) Chemotherapy 6 (38) Exemestane 6 (38)Everolimus 3 (19) Other PI3K/AKT/mTOR pathway inhibitors 4 (25)Tamoxifen 2 (13) Others 5 (31) PI3K, phosphatidylinositol 3-kinase;mTOR, mammalian target of rapamycin.

Safety:

Among 13 patients evaluable for dose-limiting toxicities (DLTs), 3 DLTswere observed, all with Compound A1 300 mg+EVE 2.5 mg+EXE 25 mg: 1 Grade3 febrile neutropenia and 2 Grade 3 alanine aminotransferase (ALT)elevation.

Hematologic AEs were the most common toxicity across all cohorts (Table5).

The most common (≥10%) Grade 3/4 study drug-related AEs were neutropenia(50%), leukopenia (31%), ALT increased (13%), and hypophosphatemia(13%).

TABLE 5 Adverse Events (All Grade >15% in All Pts) Suspected to beTreatment Related Comp Comp Comp All pts A1 A1 A1 treated Comp (200mg) + (250 mg) + (300 mg) + with A1 EVE EVE EVE Comp (600 mg) + (2.5mg) + (2.5 mg) + (2.5 mg) + A1 + EXE EXE EXE EXE EVE + (25mg) (25 mg)(25 mg) (25 mg) EXE All pts Adverse Event n = 3 n = 6 n = 1 n = 6 n = 13N = 16 Hematologic toxicities Neutropenia All 3 (100) 4 (67) 0 5 (83) 9(69) 12 (75) G¾ 2 (67) 3 (33) 0 4 (67)* 6 (46) 8 (50) ThrombocytopeniaAll 3 (100) 3 (50) 0 5 (83) 8 (62) 11 (69) G¾ 0 1 (17) 0 0 1 (8) 1 (6)Anemia All 3 (100) 2 (33) 0 5 (83) 7 (54) 10 (63) G¾ 0 0 0 0 0 0Leukopenia All 3 (100) 1 (17) 1 (100) 5 (83) 7 (54) 10 (63) G¾ 2 (67) 1(17) 0 2 (33) 3 (23) 5 (31) Lymphopenia All 0 3 (50) 0 2 (33) 5 (39) 5(31) G¾ 0 1 (17) 0 0 1 (8) 1 (6) Hypophosphatemia All 0 1 (17) 1 (100) 1(17) 3 (23) 3 (19) G¾ 0 0 1 (100) 1 (17) 2 (15) 2 (13) Non-hematologictoxicities ALT increased All 1 (33) 2 (33) 0 4 (67) 6 (46) 7 (44) G¾ 0 00 2 (33)* 2 (15) 2 (13) AST increased All 1 (33) 1 (17) 0 4 (67) 5 (39)6 (38) G¾ 0 0 0 1 (17) 1 (8) 1 (6) Stomatitis All 2 (67) 3 (50) 0 1 (17)4 (31) 6 (38) G¾ 0 0 0 0 0 0 Blood alkaline All 0 2 (33) 0 2 (33) 4 (31)4 (25) phosphatase increased G¾ 0 0 0 0 0 0 Diarrhea All 2 (67) 1 (17) 01 (17) 2 (15) 4 (25) G¾ 0 0 0 0 0 0 Nausea All 1 (33) 1 (17) 0 2 (33) 3(23) 4 (25) G¾ 0 0 0 0 0 0 Fatigue All 0 0 0 3 (50) 3 (23) 3 (19) G¾ 0 00 1 (17) 1 (8) 1 (6) Headache All 1 (33) 1 (17) 0 1 (17) 2 (15) 3 (19)G¾ 0 0 0 0 0 0 ALT, alanine aminotransferase; AST, aspartateaminotransferase; EVE, everolimus; EXE, exemestane; pt, patient; CompA1, Compound A1. *Dose-limiting toxicities included 1 Grade 3 febrileneutropenia and 2 Grade 3 ALT elevations.

Pharmacokinetics:

Mean plasma concentration-time profiles for Compound A1 and EVE inpatients treated with Compound A1+EVE+EXE on C1D15 are shown in FIGS. 28and 29.

Both Compound A1 and EVE were rapidly absorbed at steady state (C1D15);median Tmax of Compound A1 and EVE was 2 and 1 hours, respectively,across dose ranges.

At steady state, treatment with Compound A1 (200 and 300 mg)+EVE 2.5mg+EXE 25 mg resulted in Compound A1 exposure similar to that ofsingle-agent Compound A1, while EVE exposure was approximately 1.5- to2-fold and 2- to 3-fold higher than historical single-agent data whenadministered with Compound A1 200 and 300 mg, respectively.

Clinical Activity:

Of 13 patients evaluable for response, 1 patient had a confirmed partialresponse (Compound A1 300 mg+EVE 2.5 mg+EXE 25 mg), 7 patients hadstable disease (SD; Compound A1 600 mg+EXE 25 mg: 1 patient; Compound A1200 mg+EVE 2.5 mg+EXE 25 mg: 2 patients; Compound A1 300 mg+EVE 2.5mg+EXE 25 mg: 4 patients), and 1 patient had neither complete responsenor progressive disease (Compound A1 300 mg+EVE 2.5 mg+EXE 25 mg; FIG.30 and FIG. 31).

One patient with a p16 (CDKN2A) deletion, and cyclin D1 (CCND1) andinsulin-like growth factor receptor 1 (IGFR1) amplification treated withCompound A1 200 mg+EVE 2.5 mg+EXE had SD >6 months (FIG. 30).

Conclusions (Based on Interim Result)

Preliminary data suggest that the combinations of Compound A1+EXE andCompound A1+EVE+EXE are feasible, and clinical signs of activity havebeen observed in both arms of the study.

Preliminary PK analysis suggests that the 300-mg dose of Compound A1resulted in increased EVE exposure at steady state, but EVE does notaffect Compound A1 exposure.

The most common AEs were hematologic as anticipated with CDK4/6inhibitors, and were mild to moderate.

Example 6

A Phase Ib/II trial is planned. The trial will have 3 arms as describedbelow:

Arm Assigned Interventions Compound A1 Compound A1: 600 mg each day, 21days on, and 7 days off; Fulvestrant Fulvestrant: 500 mg IM Day 1 and15, followed by Q month Compound A1 Compound A1: 400 mg each day, and 21days on, 7 days off; Compound C2 Compound C2: 20 mg each day continuous;and Fulvestrant Fulvestrant: 500 mg IM Day 1 and 15, followed by Q monthCompound A1 Compound A1: 400 mg each day, 21 days on, and 7 days off;Compound C1 Compound C1: 100 mg each day continuous; and FulvestrantFulvestrant: 500 mg IM Day 1 and 15, followed by Q month

Example 7

This on-going study aims at determining antitumor efficacy of variouscompounds used as single agent, in double or in triple combination inthe HBCx-34 human breast patient-derived xenograft model.

The xenograft model proposed in this study is HBCx-34. HBCx-34 is aductal carcinoma with wild type P53, no HER2 overexpression and PR andERα overexpression. The tumor is highly responsive toadriamycine/cyclophosphamide and responsive to docetaxel andcapecitabine. HBCx-34 has got no cachexia properties, but no body weightgain is observed for HBCx-34 bearing mice.

HBCx-34 breast tumor-bearing mice will receive estrogen diluted indrinking water (β-oestradiol, 8.5 mg/l), from the date of tumor implantto the date of inclusion. No estrogen will be added during the rest ofthe study.

Female athymic nude mice (Hsd:Athymic Nude-Fox1nu), 6- to 9-week-old atthe beginning of the experimental phase, will be obtained from HarlanLaboratories (Gannat, France). Animals will be maintained in specificpathogen-free animal housing at the Center for Exploration andExperimental Functional Research (CERFE, Evry, France) animal facility.Animals will be delivered to the laboratory at least 7 days before theexperiments during which time they are acclimatized to laboratoryconditions. Mice will be housed in groups of a maximum of 7 animalsduring acclimation period and 5 animals during experimental phase. Micewill be housed inside individually ventilated cages (IVC) of Polysulfone(PSU) plastic (mm 213 W×362 D×185 H, Allentown, USA) with sterilized anddust-free bedding cobs. Food and water will be sterilized. Animals willbe housed under a light-dark cycle (14-hour circadian cycle ofartificial light) and controlled room temperature and humidity.

Compound A1: 75 Ma/Kg Free Base, p.o.

Volume of administration: 5 ml/kg (i.e. 125 μl for a 25 g mouse)Route of administration: p.o.Form: solutionVehicle: 0.5% Methylcellulose in waterConcentration: 15 mg/ml free base

Compound C2: 30 and 20 Ma/Kg Free Base, p.o.

Volume of administration: 5 ml/kg (i.e. 125 μl for a 25 g mouse)Route of administration: p.o.Form: solution

Vehicle: 10% NMP/90% PEG300

Concentration: 6 mg/ml free base=6.534 mg/ml salt base

-   -   4 mg/ml free base=4.356 mg/ml salt base        Compound C1: 35 mg/kg, p.o.        Volume of administration: 5 ml/kg (i.e. 125 μl for a 25 g mouse)        Route of administration: p.o.        Form: suspension        Vehicle: 0.5% Methylcellulose in water        Concentration: 7 mg/ml

Vehicle: NaCl 0.9%

Volume of administration: 5 ml/kg (i.e. 125 μl for a 25 g mouse)Route of administration: p.o.

Comparison Compounds: Standards of Care

Letrozole (Compound B1) 2.5 mg/kg (Femara®, Novartis)Volume of administration: 5 ml/kg (i.e. 125 μl for a 25 g mouse)Route of administration: p.o.

Form: Suspension Vehicle: 0.9% NaCl

Concentration: 0.5 mg/mlExemestane 25 mg/kg (Compound B2, Aromasine®, Pharmacia)Dose: 25 mg/kgVolume of administration: 5 ml/kg (i.e. 125 μl for a 25 g mouse)Route of administration: p.o.

Form: Suspension Vehicle: 0.9% NaCl

Concentration: 5 mg/ml

Study Groups and Regimen

1 Drug/Testing Agent 2 Drug/Testing Agent 3 Drug/Testing Agent Gr. NAgent mg/kg Route Schedule Agent mg/kg Route Schedule Agent mg/kg RouteSchedule 1 10 Vehicle — PO qd × 56* — — — — — — — — 2 10 Letrozole 2.5PO qd × 56* — — — — — — — — (Compound B1) 3 10 — — — — Compound 75 PO qd× 56* — — — — A1 4 10 — — — — — — — — Compound 30-20 PO qd × 26-30** C25 10 — — — — — — — — Compound 35 PO qd × 56*     C1 6 10 Letrozole 2.5PO qd × 56* Compound 75 PO qd × 56* — — — — (Compound A1 B1) 7 10Letrozole 2.5 PO qd × 56* — — — — Compound 30-20 PO qd × 26-30**(Compound C2 B1) 8 10 Letrozole 2.5 PO qd × 56* — — — — Compound 35 POqd × 56*     (Compound C1 B1) 9 10 Letrozole 2.5 PO qd × 56* Compound 75PO qd × 56* Compound 30-20 PO qd × 26-30** (Compound A1 C2 B1) 10 10Letrozole 2.5 PO qd × 56* Compound 75 PO qd × 56* Compound 35 PO qd ×56*     (Compound A1 C1 B1) *qd × 56: from D0 to D55 **qd × 26-30: fromD0 to D25 at 30 mg/kg then from D26 to D56 at 20 mg/kgIn combination groups, the 2 or 3 compounds will be administered withoutdelay.Dosing volume will be individually adjusted to the body weight. In eachexperimental group, the mentioned dose will be applied for all mice.

Tumorgraft Model Induction

Tumors of the same passage will be transplanted subcutaneously onto 5-10mice (donor mice, passage (n−1)). When these tumors reach 1000 to 2000mm³, donor mice will be sacrificed by cervical dislocation, tumors willbe aseptically excised and dissected. After removing necrotic areas,tumors will be cut into fragments measuring approximately 20 mm³ andtransferred in culture medium before grafting.

Mice will be anaesthetized with ketamine/xylazine, and then skin will beaseptized with a chlorhexidine solution, incised at the level of theinterscapular region, and a 20 mm³ tumor fragment will be placed in thesubcutaneous tissue. Skin will be closed with clips.

All mice from the same experiment will be implanted on the same day.

Inclusion Criteria

Healthy mice aged 6 to 9 weeks and weighing at least 20 g will beincluded in the study. Mice will be allocated to different groupsaccording to their tumor volume to give homogenous mean and median tumorvolume in each treatment arms. Treatments will be randomly attributed tocages housing up to 5 mice.

For each group, 10 mice with established tumors and average tumor volumeranging 108 (6×6) to 288 (9×8) mm³ will be included in the study. In thecase that tumor growth is heterogeneous, group size may be reduced (upto 8 mice/group) and/or inclusion may be staggered.

Animals Observations

From grafting day to study termination, animals will be observed everyday, for physical appearance, behavior and clinical changes.

Tumor Measurements and Body Weight Monitoring

Tumor volume will be evaluated by measuring tumor diameters, with acalliper, biweekly during the treatment period and once a week duringthe follow-up period. The formula TV (mm³)=[length (mm)×width (mm)²]/2will be used, where the length and the width are the longest and theshortest diameters of the tumor, respectively.

Tumors will not be weighed at the end of experimental phase.

All animals will be weighted biweekly during the treatment period andonce a week during the follow-up period.

Unless specified otherwise by the Sponsor, in case that body weight lossreaches 15% compared to the 1^(st) day of treatment, DietGel Recovery®will be given for the entire group in which the body weight loss isobserved.

Criteria for Ethical Sacrifice

Each animal will be sacrificed if one of the following conditions ismet:

-   -   Body weight loss (BWL) ≥20% compared to the 1st day of treatment        for 3 consecutive measurements (2 days or 48 hours).    -   General alteration of behaviour or clinical signs.    -   Tumor volume ≥2000 mm³.

Unless specified otherwise, no necropsy will be performed at sacrifice.

End Points (Whichever Comes First)

Each group of animals will be sacrificed if the two following conditionsare met:

-   -   A tumor volume of 2000 mm³ is reached for at least one animal    -   And the initial median tumor volume has been increased by 3 to        5-fold.

The endpoints for the experiment are:

-   -   a treatment phase of 8 weeks*    -   and a follow-up phase of 57 days.        (*) Treatment phase could be extended by 2 or 3 weeks if no        toxicity is observed and if required according to “Tumorgraft        model induction”.

Data Analysis

Day 0 will be always considered the first day of treatment. The days ofthe experiment will be subsequently numbered according to thisdefinition. Recordings will be expressed as mean+/−standard error of themean (mean+/−sem) and median+/−interquartile (median+/−IQR).

Statistical analysis will be done for each measurement by Mann-Whitneynon parametric comparison test using GraphPad Prism software. Eachtreated group will be compared with control group.

FIGS. 20-22 illustrates some results of this study.

1.-29. (canceled)
 30. A pharmaceutical combination comprising (1) afirst agent which is Compound A1 described by Formula A1 below or apharmaceutically acceptable salt thereof:

and (2) a second agent which is fulvestrant. 31.-42. (canceled)
 43. Apharmaceutical combination consisting essentially of (1) a first agentwhich is Compound A1 described by Formula A1 below or a pharmaceuticallyacceptable salt thereof:

and (2) a second agent which is letrozole.
 44. The combination of claim43, wherein the agents are administered simultaneously, separately orsequentially.
 45. The combination of claim 43, wherein the agents areadministered simultaneously.
 46. The combination of claim 43, whereinthe agents are administered separately.
 47. The combination of claim 43,wherein the agents are sequentially.
 48. A method of palliativelytreating breast cancer consisting essentially of administering to asubject (1) a first agent which is Compound A1 described by Formula A1below or a pharmaceutically acceptable salt thereof:

and (2) a second agent which is letrozole.
 49. The method of claim 48,wherein the breast cancer is an estrogen receptor positive breastcancer.
 50. The method of claim 48 wherein the breast cancer is a HER2−breast cancer.
 51. The method of claim 48, wherein the breast cancer isan estrogen receptor positive, HER2− breast cancer.
 52. The method ofclaim 48, wherein the breast cancer is a hormone receptor positive,HER2− breast cancer.
 53. The method of claim 48, wherein the agents areadministered simultaneously.
 54. The method of claim 48, wherein theagents are administered separately.
 55. The method of claim 48, whereinthe agents are sequentially.
 56. The method of claim 48, wherein thebreast cancer is an advanced breast cancer.
 57. The method of claim 56,wherein the breast cancer is an estrogen receptor positive, HER2−advanced breast cancer.
 58. The method of claim 48, wherein the breastcancer is a metastatic breast cancer.
 59. The method of claim 58,wherein the breast cancer is an estrogen receptor positive, HER2−metastatic breast cancer.